Rehydratable pharmaceutical product

ABSTRACT

A pharmaceutical product comprising lyophilised polymer matrix including a biologically active compound, of particular utility for embolisation, having improved rehydration properties is packaged in an airtight package under vacuum.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/305,048, filed Jan. 8, 2009, now allowed, which is a national stageentry of PCT/EP2007/056282, filed Jun. 22, 2007, which claims priorityto EP application No. 0625324.9 filed Jun. 22, 2006. The entiredisclosures of the prior applications are considered part of thedisclosure of the accompanying continuation application, and are herebyincorporated by reference.

BACKGROUND OF THE INVENTION

The present invention relates to methods for formulating storage stableand easily rehydratable dried pharmaceutical compositions foradministration to animals, especially for use as chemo-emboliccompositions.

Embolisation therapy involves the introduction of an agent into thevasculature in order to bring about the deliberate blockage of aparticular vessel. This type of therapy is particularly useful forblocking abnormal connections between arteries and veins, such asarteriovenous malformations (AVMs), and also for occluding vessels thatfeed certain hyper-vascularised tumours, in order to starve the abnormaltissue and bring about its necrosis and shrinkage. Examples of areas inwhich embolotherapy is increasingly being used are for the treatment ofmalignant hyper-vascular tumours such as hepatocellular carcinoma (HCC)and the treatment of uterine fibroids.

In the case of HCC it may be desirable to treat the tumour with anembolisation agent loaded with a chemotherapeutic agent. DC bead is anembolisation bead that can be loaded with doxorubicin prior toadministration to a patient. It may be more convenient, however, if thebeads could be supplied to the interventional radiologist with thedoxorubicin already pre-loaded into the embolic agents. This saves timein preparation, handling of toxic drug and also the need to guess theamount of agent required for the procedure.

Due to the fact that many drugs, such as doxorubicin, are potentiallyunstable over time when in the hydrated form, an embolic adduct withpre-loaded drug may be lyophilised or freeze dried to remove excesswater prior to terminal sterilization. The lyophilisation processresults in the formation of a free flowing dry powder which isrelatively stable during storage. This product is described in WO2004/071495 A1.

In WO 2004/071495 A1 the polymer matrix is a cross-linked polyvinylalcohol. Other particulate embolic materials are available, for instancebased on alginates, albumin, gelatin, other synthetic polymers includingPVA cross-linked with aldehydes, polyacrylates, polylactic- andpolyglycolic acids. These may be in the form of irregular particles or,preferably, microspheres.

A number of other simple therapeutic compounds are being investigated incombination with microspheres for the embolization of other tumourtypes. Examples include irinotecan (WO-A-2006027567) and ibuprofen(WO-A-2006013376). In addition, newer drugs are becoming more complex instructure and there is a move away from simple molecular entities tomuch more complex entities which in some cases are of biological origin.These more complex molecular entities will probably be more unstablethan corresponding simple molecular entities so the need for freezedrying of microspheres loaded with these species will probably berequired to prolong their shelf-life.

One problem with freeze drying gels, e.g. hydrogels, or macroporousmicrospheres is that air pockets develop within the microspheres as thewater is removed during the drying process. We have identified the factthat the presence of these air pockets is problematic when the dry beadsare rehydrated. They can hinder the rate of hydration of the beads sincethe air needs to be exchanged with liquid for the bead to be fullyhydrated. Since air is relatively hydrophobic and the aqueous liquidsused for rehydration of the microspheres are hydrophilic, this processcan be slow. In some cases, we have found hydration is totally inhibitedby the presence of the air pockets within the micro spheres. One otherconsequence of the presence of trapped air inside the microspheres isthe buoyancy of the microspheres is altered. Since the air is less densethan the liquid for rehydration the beads tend to float. This can bevery problematic and can affect the potential to obtain an adequatesuspension of the beads when hydrated, in for instance, a mixture ofwater and contrast agent. In order to deliver the microspheres anadequate suspension is required in the hydration medium for sufficienttime to allow ease of handling and effective delivery through amicro-catheter. Homogeneous delivery of microspheres andsuspending/contrast medium allows control of the dose of microspheresand of active.

SUMMARY OF THE INVENTION

The present invention overcomes these problems of speed of hydration andineffective suspension and avoids the addition of additional excipientsto the particles.

According to the invention there is provided a new method forformulating the dried product suitable for direct administration into ananimal after rehydration to form a suspension comprising:

-   -   i) a freezing step in which particles of polymer matrix swollen        with water and having absorbed therein a non-volatile        biologically active compound are cooled to a temperature below        the freezing point of water;    -   ii) a lyophilisation step in which the cooled particles from        step i) are subjected to a reduced pressure at which ice        sublimes for a period during which at least a portion of the        absorbed ice sublimes and water vapour is removed to form dried        particles; and    -   iii) a packaging step in which the dried particles are packaged;    -   characterised in that the packaging step is carried out under        reduced pressure and the package containing the particles is        substantially airtight and has an interior under vacuum.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C, 1D and 1E are diagrammatic representations of theapparatus in which the examples are carried out showing how the vialsare stopped without allowing ingress of air.

DETAILED DESCRIPTION OF THE INVENTION

The steps i) and ii) are generally carried out under the same conditionsas in general lyophilisation processes for pharmaceuticals. It isconvenient for further drying steps to be included, for instance betweensteps ii) and iii). Such further drying steps may be carried out toremove additional water and may be carried out at a temperature abovethe freezing point of water and at a reduced pressure, for instance at apressure lower than the pressure for which step i) is conducted.Suitable cycles are known comprising a combination of cold low pressuresteps, followed by warmer, further reduced pressure steps, wherebywater, including physically bound water, is removed from the polymermatrix.

Suitable pressures under which the lyophilisation step is carried outare in the range 0.01 mbar to 0.1 bar, preferably less than 100 mbaroften less than 10 mbar, e.g. less than 1 mbar often 0.02 mbar andupwards. Suitable temperatures for the cooling and lyophilisation stepare less than −10° C., preferably less than −15° C., often less than−20° C., for instance down to −50° C., preferably around −30° C.

Suitable pressures at which a further drying step between steps ii) andiii), are less than 0.2 mbar, preferably less than 0.1 bar, for instancedown to 0.01 mbar, preferably about 0.05 mbar. Suitable temperatures areat least 0° C., preferably at least 25° C., more preferably at least 60°C.

Generally the temperatures and pressures are to be adapted dependantupon the volumes, especially the depths of the container of beads beingtreated. Shallow containers generally require less time to be frozen andshorter low pressure cycles than deep containers containing largequantities of materials. The freezing step may be carried out for aperiod of at least 5 minutes, for instance at least 10 minutes often anhour or more. The lyophilisation step may be carried out for a period ofat least one hour, often overnight, for instance for a period of atleast 8 hours or even more. The further drying step may be carried outover a period of at least an hour preferably two hours or more.

Although it is possible to carry out the method of the invention inbulk, with subsequent weighing and packaging in dosage forms, allcarried out under vacuum, it is most convenient for steps i) and ii) orthe method of the invention to be carried out with the swollen particlesalready contained within the vessels in which they are finally to bepackaged. The vessels thus each contain a single dose of particles withbiologically active compound. In such processes, the vessels arepreferably formed of a rigid material, and have a mouth which isstoppered in the packaging step using a suitable airtight stopper.Suitable vessels are formed of glass, or may be rigid airtight plasticswhich are physically stable at the temperatures to which the material issubjected during the method of the invention. Most conveniently themethod of the invention is carried out in an apparatus which is capablesimultaneously of carrying out the freeze-drying and stoppering steps.Preferably substantially without allowing ingress air, oxygen or othergas after step ii) and before step iii). A suitable apparatus iscommercially available under the trade name Epsilon 1-6D freeze drier byChrist and Genesis freeze drier by VirTis. Stoppers which are adequatelyairtight over useful storage periods are made of, for instance, butylrubber although other low permeability rubbers which are stable attemperatures down to −30 or less ° C., may be used. It is particularlyconvenient for the stoppers to be formed of a material which may bepierced with a hypodermic needle, so that rehydrating liquid may beeasily injected into the stoppered vessels. During storage the pressureinside the vessel may increase as air slowly permeates through thestopper, but the permeability should be such that the pressure insidethe vessel is less than atmospheric after storage periods of at least amonth, preferably at least a year, for instance two years or more.

The method of the invention is particularly suitable for formulatingcompositions in which the polymer is a water-insoluble, preferably butnot limited to a substantially non-biodegradable, pharmaceuticallyacceptable polymer. Since the starting material is water-swollen, thenthe polymer must be water-swellable. At the start of the method of theinvention the polymer is preferably swollen substantially to equilibriumin aqueous liquid. Generally there is substantially no extra-particulateaqueous liquid, and the method may involve a preliminary step in which asuspension of swollen polymer particles in an aqueous liquid issubjected to an initial drying step in which extra particulate liquid isremoved, for instance by decantation, filtration or centrifugation.

The polymer is preferably cross-linked, most preferably covalentlycross-linked, although ionically cross-linked polymers may also beusefully formulated using the method of the invention. Ionicallycross-linked materials may comprise, for instance, ionically chargedpolymer, cross-linked with counterionically charged second polymer or,alternatively, with multivalent metal ions.

It may be suitable to use polymers which are derived from naturalsources, such as albumin, alginate, gelatin, starch, chitosan orcollagen, all of which have been used as embolic agents. Naturalpolymers or derivatives may be combined with synthetic polymers, byblending, inter molecular cross-linking or grafting. However, preferablythe polymer is preferably substantially free of naturally occurringpolymer or derivatives.

Preferably the polymer is based on a synthetic material, for instanceformed by polymerisation of ethylenically unsaturated monomer,preferably in the presence of cross-linking monomer, for instancemacromer or di- or higher-functional cross-linking monomers.

The ethylenically unsaturated monomers may include an ionic (includingzwitterionic) monomer.

Copolymers of hydroxyethyl methacrylate, acrylic acid and cross-linkingmonomer, such as ethylene glycol dimethacrylate or methylenebisacrylamide, as used for etafilcon A based contact lenses may be used.Copolymers of N-acryloyl-2-amino-2-hydroxymethyl-propane-1,3-diol andN,N-bisacrylamide may also be used.

Other suitable polymers are cross-linked styrenic polymers, e.g. withionic substituents, of the type used as separation media or as ionexchange media, and polyphosphazenes.

Another type of polymer which may be used to form the water-swellablewater-insoluble matrix is polyvinyl alcohol cross-linked using aldehydetype cross-linking agents such as glutaraldehyde. For such products, thepolyvinyl alcohol (PVA) may be rendered ionic or may be substantiallynon-ionic. For instance the PVA may be rendered ionic by providingpendant ionic groups by reacting a functional ionic group containingcompound with the hydroxyl groups. Examples of suitable functionalgroups for reaction with the hydroxyl groups are acylating agents, suchas carboxylic acids or derivatives thereof, or other acidic groups whichmay form esters. Suitable commercially available embolic agents based onpolyvinyl alcohol which may be used in the invention are Ivalon™,Trufill®, Contour SE™ and Hepasphere™.

The invention is of particular value where the polymer matrix is formedof a polyvinyl alcohol macromer, having more than one ethylenicallyunsaturated pendant group per molecule, by radical polymerisation of theethylenic groups. Preferably the PVA macromer is copolymerised withethylenically unsaturated monomers for instance including a nonionicand/or ionic monomer.

The PVA macromer may be formed, for instance, by providing PVA polymer,of a suitable molecular weight such as in the range 1,000 to 500,000 D,preferably 10,000 to 100,000 D, with terminal or mid-chain pendantvinylic or acrylic groups. Pendant acrylic groups may be provided, forinstance, by reacting acrylic or methacrylic acid with PVA to form esterlinkages through some of the hydroxyl groups. Other methods forattaching vinylic groups capable of polymerisation onto polyvinylalcohol are described in, for instance, U.S. Pat. No. 4,978,713 and,preferably, U.S. Pat. Nos. 5,508,317 and 5,583,163. Thus the preferredmacromer comprises a backbone of polyvinyl alcohol to which is linked,via a cyclic acetal linkage, an (alk)acrylaminoalkyl moiety. Example 1of WO 2004/071495 describes the synthesis of an example of such amacromer known by the approved named nelfilcon B which is useful in thisinvention. Preferably the PVA macromers have about 2 to 20 pendantethylenic groups per molecule, for instance 5 to 10.

Where PVA macromers are copolymerised with ethylenically unsaturatedmonomers including an ionic monomer, the ionic monomer preferably hasthe general formula I

Y¹BQ

in which Y¹ is selected from

-   CH₂═C(R)—CH₂—O—, CH₂═C(R)—CH₂ OC(O)—, CH₂═C(R)OC(O)—, CH₂═C(R)—O—,-   CH₂═C(R)CH₂OC(O)N(R¹)—, R²OOCCR═CRC(O)—O—, RCH═CHC(O)O—,-   RCH═C(COOR²)CH₂—C(O)—O—,

wherein:

R is hydrogen or a C₁-C₄ alkyl group;

R¹ is a hydrogen or a C₁-C₄ alkyl group;

R² is a hydrogen or a C₁-C₄ alkyl group or BQ where B and Q are asdefined below;

A is —O— or —NR¹—;

K¹ is a group —(CH₂),OC(O)—, —(CH₂),C(O)O—, —(CH₂),OC(O)O—,—(CH₂)_(r)NR³—, —(CH₂)_(r)NR³C(O)—, —(CH₂)_(r)C(O)NR³—,—(CH₂)_(r)NR³C(O)O—, —(CH₂)_(r)OC(O)NR³—, —(CH₂)_(r)NR³C(O)NR³— (inwhich the groups R³ are the same or different), —(CH₂)_(r)O—,—(CH₂)_(r)SO³—, or, optionally in combination with B¹, a valence bondand r is from 1 to 12 and R³ is hydrogen or a C₁-C₄ alkyl group;

B is a straight or branched alkanediyl, oxaalkylene,alkanediyloxaalkanediyl, or alkanediyloligo (oxaalkanediyl) chainoptionally containing one or more fluorine atoms up to and includingperfluorinated chains or, if Q or Y¹ contains a terminal carbon atombonded to B a valence bond; and

Q is an ionic group.

An anionic group Q may be, for instance, a carboxylate, carbonate,sulphonate, sulphate, nitrate, phosphonate or phosphate group. Themonomer may be polymerised as the free acid or in salt form. Preferablythe pK_(a) of the conjugate acid is less than 5.

A suitable cationic group Q is preferably a group N⁺R⁴ ₃, P⁺R⁵ ₃ or S⁺R⁵₂ in which the groups R⁴ are the same or different and are eachhydrogen, C₁-C₄-alkyl or aryl (preferably phenyl) or two of the groupsR⁴ together with the heteroatom to which they are attached from asaturated or unsaturated heterocyclic ring containing from 5 to 7 atomsthe groups R⁵ are each OR⁴ or R⁴. Preferably the cationic group ispermanently cationic, that is each R⁴ is other than hydrogen. Preferablya cationic group Q is N⁺R⁴ ₃ in which each R⁴ is C₁-C₄-alkyl, preferablymethyl.

A zwitterionic group Q may have an overall charge, for instance byhaving a divalent centre of anionic charge and monovalent centre ofcationic charge or vice-versa or by having two centres of cationiccharge and one centre of anionic charge or vice-versa. Preferably,however, the zwitterion has no overall charge and most preferably has acentre of monovalent cationic charge and a centre of monovalent anioniccharge.

Examples of zwitterionic groups which may be used as Q in the presentinvention are disclosed in WO 00/29481 A1.

Where the ethylenically unsaturated monomer includes zwitterionicmonomer, for instance, this may increase the hydrophilicity, lubricity,biocompatibility and/or haemocompatibility of the particles. Suitablezwitterionic monomers are described in our earlier publications WO92/07885 A, WO 94/16748 A, WO 94/16749 A and WO 95/20407 A. Preferably azwitterionic monomer is 2-methacryloyloxy-2′-trimethylammonium ethylphosphate inner salt (MPC).

In the monomer of general formula I preferably Y¹ is a group CH₂═CRCOA—in which R is H or methyl, preferably methyl, and in which A ispreferably NH. B is preferably an alkanediyl group of 1 to 12,preferably 2 to 6 carbon atoms. Such monomers are acrylic monomers.

There may be included in the ethylenically unsaturated monomer diluentmonomer, for instance non-ionic monomer. Such a monomer may be useful tocontrol the pK_(a) of the acid groups, to control the hydrophilicity orhydrophobicity of the product, to provide hydrophobic regions in thepolymer, or merely to act as inert diluent. Examples of non-ionicdiluent monomer are, for instance, alkyl (alk) acrylates and (alk)acrylamides, especially such compounds having alkyl groups with 1 to 12carbon atoms, hydroxy, and di-hydroxy-substituted alkyl(alk) acrylatesand -(alk) acrylamides, vinyl lactams, styrene and other aromaticmonomers.

In the polymer matrix, where there is ionic group present the level ofion is preferably in the range 0.1 to 10 meq g⁻¹, preferably at least1.0 meq g⁻¹.

Where PVA macromer is copolymerised with other ethylenically unsaturatedmonomers, the weight ratio of PVA macromer to other monomer ispreferably in the range of 50:1 to 1:5, more preferably in the range20:1 to 1:2. In the ethylenically unsaturated monomer the ionic monomeris preferably present in an amount in the range 10 to 100 mole %,preferably at least 25 mole %.

The polymer may be formed into particles in several ways. For instance,the cross-linked polymer may be made as a bulk material, for instance inthe form of a sheet or a block, and subsequently be comminuted to thedesired size. Alternatively, the cross-linked polymer may be formed assuch in particulate form, for instance by polymerising in droplets ofmonomer in a dispersed phase in a continuous immiscible carrier.Examples of suitable water-in-oil polymerisations to produce particleshaving the desired size, when swollen, are known. For instance U.S. Pat.No. 4,224,427 describes processes for forming uniform spherical beads(microspheres) of up to 5 mm in diameter, by dispersing water-solublemonomers into a continuous solvent phase, in a presence of suspendingagents. Stabilisers and surfactants may be present to provide controlover the size of the dispersed phase particles. After polymerisation,the cross-linked microspheres are recovered by known means, and washedand optionally sterilised. Preferably the particles, e.g. microspheres,are swollen in an aqueous liquid, and classified according to theirsize.

The method of the invention is of particular utility where therehydrated material is to be used as an embolic agent. The particles arepreferably microspheres, that is are formed of substantially sphericalor spheroidal particles. For embolic agents, such particles aregenerally separated into size fractions, whereby a surgeon may selectmicrospheres of a size suited to embolise the vessels desired to beblocked in the method of treatment. For embolic materials, the particlesgenerally have average diameter in the range 40 to 2000 μm, morepreferably in the range 100 to 1500 μm. Preferably the particles havesizes such that they fall in a nominal range around 200 μm to 300 μm inwidth. Suitable size fractions have nominal sizes in the range 100 to300, 300 to 500, 500 to 700, 700 to 900, 900 to 1200 μm. The particlesizes may be determined in the invention at various stages of themethod. For instance the particle sizes may be determined on the swollenparticles used as the starting materials for step i). Alternatively theparticle sizes may be measured on the rehydrated products of the methodof the invention.

Preferably the method of the invention comprises preliminary loadingsteps in which particles of non-loaded polymer are loaded with thebiologically active compound. In such a method, the non-loaded polymerparticles generally have sizes in the range defined above, when swollento equilibrium at room temperature, in 0.9 wt % NaCl. The invention, asindicated above, is of particular value where the particles are to berehydrated to form an aqueous suspension which is ultimately to be usedas by direct administration to an animal, for instance by directinjection into a tumour or other target for local administration of theactive. A preferred use is as an embolic material. Of particularutility, embolic materials are utilised to embolise solid tumours,particularly malignant tumours although they may also be of use toembolise benign tumours such as uterine fibroids. Biologically activematerials are preferably anti-tumour compounds, especially compoundswhich are unstable in the presence of water or other solvents. Theinvention is of particular value where the biologically active compoundis an anti-neoplastic or often anti-proliferation, anti-migratory,immunosuppressant, analgesic, anti-inflammatory, anti-pyretic,anti-bacterial or anaesthetic agent.

The invention is of particular value for formulating anti-neoplasticsand immunosuppressants, such as angiopeptin, and statins, such assandostatin. Other suitable drugs include azacitidine, bleomycin andbleomycin sulphate, carboplatin, cisplatin, streptozoticin,capecitabine, vinorelbine, cyclosporin, cytabanine, decarbazine,anthracyclines such as daunorubicin, doxorubicin, epirubicin,mitoxantrone and banoxantrone. Other suitable chemotherapeutics includefluorouracil, gemcitabine, ifosfamide, methotrexate, mitomycin, mustinehydrochloride, lomustine, carmustine/BCNU, meclorethamine, vincristine,vinblastine, cytosar/cytarabine, peclitaxel, docetaxel, rapamycin andderivatives, such as tacrolimus, everolimus, biolimus, zotarolimus, andRAD001. Other suitable drugs include tyrphostin, tetradecylselenoaceticacid, tetradecylthioacetic acid, ethylisopropylamiloride, antithrombin,aggrastat, cilostazol, clexane, clopidogrel, dipyridamole, persantine,integrillin, abciximab, trapidil, VEGF, carredilol, estradiol and otherestrogens, L-arginine, nitric oxide donors, probucol, quinaprilat,thioctacid, telmisartan, zoledronate and matrix metalloproteinaseinhibitors such as batimastat and marimastat.

Another class of compounds for which the invention is of utility includeanalgesics, anti-inflammatories, and anti-pyretics, such as Codeinesulphate, Diamorphine hydrochloride, fentanyl, hydromorphonehydrochloride, indomethacin, morphine hydrochloride and pethidinehydrochloride.

The invention may also be of utility for formulating anti-bacterials,for instance which may be administered into the arteriovenous systemand/or may be administered in extended/controlled release formulationscomprising polymer matrix formulations. Examples of such anti-bacterialsare ampicillin, benzyl-penicillin, ceftazidime, ceftriaxone sodium,gemtamicin sulphate, tetracycline and vancomycin hydrochloride.

The method of loading the biologically active compound into the polymermatrix to form the starting material for step i) of the invention isselected according to the solubility of the active in solventscompatible with the polymer matrix and/or the swellability of thepolymer in such solvents. For instance, in one preferred combination ofcomponents, the polymer is generally ionically charged, and is loaded byan ion-exchange type process with counterionically charged activecompound. Where the active is doxorubicin hydrochloride, for instance,which is cationically charged, the polymer matrix is preferablyanionically charged.

According to a preferred aspect of the invention the therapeutic activeused in the present invention is an anthracycline compound, whichcomprises an anthraquinone group to which is attached an amine sugar.The amino group on the sugar is believed to associate with anionicgroups in the polymer matrix, to allow high levels of loading andcontrolled delivery after administration. Alternatively the amine groupscan be pendant groups on the anthracycline ring as for mitoxantrone andbanoxantrone.

Examples of suitable anthracyclines have the general formula II

Further a polymer matrix which allows good loading levels and release isan anionic poly (vinyl alcohol) based material, preferably formed bycopolymerising the PVA macromer described above with an ionic monomer ofthe general formula I in which Q is an anionic group whose conjugateacid preferably has a pK_(a) of 5 or less.

We have found that doxorubicin, which has been thoroughly tested forefficacy on various tumours, has particularly interesting loading andrelease characteristics. The drug appears to have a particular affinityfor poly(vinyl alcohol-graft-acrylamido propane sulphonic acid), so thathigh levels of doxorubicin are capable of incorporation into thepolymer, and release over many days.

According to another preferred embodiment the pharmaceutical active is acamptothecin preferably a cationically charged camptothecin used incombination with an ionically charged polymer. Examples of suchcamptothecins have general formula III

in which R¹⁰ is H, lower C₍₁₋₁₆₎alkyl, optionally substituted by ahydroxyl amine, alkoxy, halogen, acyl or acyloxy group or halogen; and

R⁹ is chlorine or NR¹¹R¹² where R¹¹ and R¹² are the same or differentand each represent a hydrogen atom, a substituted or unsubstituted C₁₋₄alkyl group or a substituted or unsubstituted carbocyclic orheterocyclic group, or R¹¹ and R¹² together with the nitrogen atom towhich they are attached form an optionally substituted heterocyclic ringwhich may be interrupted by —O—, —S— or >NR¹³ in which R¹³ is a hydrogenatom, a substituted or unsubstituted C₁₋₄ alkyl group or a substitutedor unsubstituted phenyl group;

and wherein the grouping —O—CO—R⁹ is bonded to a carbon atom located inany of the 9, 10 or 11 positions in the A ring of the camptothecincompound, including salts thereof.

Preferably R⁹ is NR¹¹ R¹² in which R¹¹ and R¹² together with thenitrogen atom from an optionally substituted heterocyclic ring. Mostpreferably R⁹ is

Preferably R⁹ is substituted at the 10 position in the camptothecin.Preferably R¹⁰ is ethyl.

The therapeutic active may be incorporated into the polymer matrix by avariety of techniques. In one method, the therapeutic active may bemixed with a precursor of the polymer, for instance a monomer ormacromer mixture or a cross-linkable polymer and cross-linker mixture,prior to polymerising or cross-linking. Alternatively, the active may beloaded into the polymer after it has been cross-linked. For instance,particulate dried polymer may be swollen in a solution of therapeuticactive, preferably in water, optionally with subsequent removal ofnon-absorbed agent and/or evaporation of solvent. A solution of theactive, in an organic solvent such as an alcohol, or, more preferably,in water, may be sprayed onto a moving bed of particles, whereby drug isabsorbed into the body of the particles with simultaneous solventremoval. Most conveniently, we have found that it is possible merely tocontact swollen particles suspended in a continuous liquid vehicle, suchas water, with a solution of drug, over an extended period, whereby drugbecomes absorbed into the body of the particles. This is believed to beanalogous to a cation exchange type process. The swelling vehicle maysubsequently be removed or, conveniently, may be retained with theparticles as part of the product for subsequent use as an embolic agent.

The drug loaded particles are then recovered from excess loadingsolution or solvent and subjected to the above described drying andpackaging process.

The present invention comprises further a method preparing apharmaceutically acceptable suspension for administration to an animalin which the product of the method of the invention defined above isrehydrated by adding to the package of dried product a pharmaceuticallyacceptable sterile aqueous liquid and, optionally, a contrast medium, toform a suspension of swollen particles in a continuous aqueous liquid.

The pharmaceutically acceptable sterile aqueous liquid is, for instance,physiological saline, deionised water or, preferably, phosphate bufferedsaline. Preferably the sterile aqueous liquid is added directly into theairtight package by puncturing this with a hypodermic needle throughwhich the liquid is directed without allowing ingress of gases, such asair or oxygen. Once the aqueous rehydrating liquid and particles haveformed a stable suspension, this is preferably combined with contrastmedium and mixed, to form a suspension ready for administration to apatient.

There is also provided in the invention a method of treatment of ananimal in which the suspension formed in the preceding paragraph isadministered to an animal, preferably by administration into an arteryto embolise blood vessels, preferably to embolise a solid tumour.

According to the invention there is also provided a new airtight packagecontaining, under vacuum, lyophilised particles of water-swellablewater-insoluble biocompatible polymer into which is absorbed apharmaceutically acceptable biologically active compound, in which theparticles are swellable in 0.9 wt % saline at room temperature to sizesin the range 40 to 2000 μm.

In this aspect of the invention the polymer and biologically activecompound have the preferred properties defined above in connection withthe first aspect of the invention.

The invention is illustrated further in the accompanying examples.

Example 1 Microsphere Production

The spheres are synthesised by a method of suspension polymerisation inwhich an aqueous phase comprising a solution (about 700 g) containing aPVA macromer, nelfilicon A, (around 80 g), 2-acrylamido-2-methyl propanesulphonate sodium salt (70 g) and potassium persulphate initiator(around 5 g) is suspended in an organic phase of butyl acetate (31) and5 g cellulose acetate butyrate (solution in ethyl acetate) in a stirredreactor. By employing rapid mixing the aqueous phase can be dispersed toform droplets, the size and stability of which can be controlled byfactors such as stirring rates, viscosity, ratio of aqueous/organicphase. Polymerisation of the dispersed monomer/macromer solution isinitiated by the addition of TMEDA and raising the temperature to over50° C. for several hours under nitrogen. After cooling to roomtemperature the product is purified by removing the butyl acetate byfiltration followed by washing steps with solvents, vacuum dried toremove solvents then the microspheres are equilibrated at 60° C. inwater to fully re-hydrate. The spheres are sieved using a 316 Lstainless steel vortisieve unit (MM Industries, Salem Ohio) withstainless steel sieving trays with mesh sizes ranging from 32 to 1200 μmincluding sizes about 100 μm, 300 μm, 500 μm, 700 μm and 900 μm. Spherescollected in the 32 μm sieve are discarded.

Drug Loading

For each size of microsphere used, 0.5 ml was transferred in to 2, 1 mlsyringes, one for drug take up and the second to act as a control. Thesizes chosen for the experiment were, 100-300 μm, 300-500 μm, 500-700 μmand 850-1000 μm. Additionally a further 3 syringes of the 500-700 μmwere prepared in order to validate the procedure. 11, 10 ml glass vialswere covered in foil, to prevent degradation of the doxorubicin by lightfor the duration of the experiment. A standard curve was created. Usingthe 80 ml, 20 mg/ml drug solution, the following concentrations wereprepared and their absorbances (at 483 nm) measured: 100 μg/ml, 50μg/ml, 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml and 3.125 μg/ml. The resultingabsorbances were plotted on a graph and the equation of the line used tocalculate the concentration of drug that was uptaken by the beads in theexperiment. Four of the vials were filled with 5 ml of distilled water(ROMIL) to be used as controls when the beads were added. To theremaining 7 vials were added 5 ml of the drug solution at the desiredconcentration. The starting absorbance and therefore concentration ofthe solution was already known from the preparation of the standardcurve. (In order to measure the absorbance of the 20 5 mg/ml solution itwas necessary to dilute it 200 times, using the concentration 100 μg/ml.This 1:200 dilution was carried through for the duration of measuringthe uptake of the solution by the beads.) The stopwatch was started assoon as the first set of microspheres were added to the first drugcontaining vial, microspheres were added to each of the remaining 6vials working from smallest to largest. Once sealed using the caps theywere placed on the rotary mixer. The process was repeated for thecontrol samples. The absorbances were measured in the same order as thevials were set up at time intervals of 0.167 hr (10 min), 0.5 hr, 1 hr,2 hr, 24 hrs and 96 hrs. From the data the amount of drug (in mg) per 1ml of microspheres and the % uptake of drug by 1 ml of microspherescould be calculated.

Epsilon 1-6D freeze dryer is used in the next step of the production of100-300, 300-500, 500-700 and 700-900 μm (1.5±0.1 ml) preloaded with37.5 mg doxorubicin per vial. The vials are type 1 tubular neutral glass10 ml vials and the bungs are butyl rubber igloo lyophilisationstoppers. The lyophilisation program has been designed to operate whenfully loaded with 345 vials of microspheres.

Epsilon 1-6D freeze dryer with Lyo Screen Control (LSC) panel andPfeiffer DUO 10 Rotary Vane Vacuum pump. The apparatus is controlled byLyolog LL-1 documentation software.

The Epsilon 1-6D is a pilot scale freeze dryer. The system consists of achamber with three liquid controlled cooled/heated shelves with atemperature range of −40 to 80° C. on which samples are frozen. The icecondenser, whose minimum temperature is −60° C., is located in anadjacent chamber separated by an intermediate valve. The shelves and icecondenser are cooled using two cooling machines. The chamber pressure isachieved using a Pfeiffer DUO 10 rotary vane vacuum pump.

The Epsilon 1-6D can lyophilise a maximum of 345 vials (10 ml) percycle, i.e. with 115 vials per shelf.

The microspheres are lyophilised by freezing at about −30° C. without avacuum, at least 1 h, then reducing the pressure gradually over a periodof about half an hour to a pressure of in the range 0.35-0.40 mbar,while allowing the temperature to rise to about −20° C. and holding theconditions at this temperature and pressure overnight, followed byraising the temperature to room temperature for a period of about 1-2hours at the same time pressure, followed by a period at roomtemperature with the pressure reduced to about 0.05 mbar, to a totalcycle time of 24 hours.

At the end of the cycle and substantially without allowing ingress ofair the vials are stoppered under vacuum by turning the vial closingmechanism that lowers the shelves to stopper the vials on the shelfbeneath. The chamber is then aerated to allow the chamber to reachatmospheric pressure. The shelves are then returned to their originalposition and the chamber opened.

As a control, the process is repeated but the chamber is allowed toequilibrate to atmospheric pressure by allowing ingress of air toatmospheric pressure prior to the vials being stoppered.

The products of the method of the invention loaded with doxorubicin(typically 25 to 40 mg drug per ml bead) and of the comparison methodare then rehydrated by injection of 3 ml of water and 3 ml contrastagent (e.g. Lipiodiol) using a conventional hypodermic needle attachedto a syringe to pierce the stopper. The vials are shaken for 3 minutesmanually or using a mechanical shaker. The method of the invention thusallows faster rehydration and easier handling both of which enable morecontrol of dosage which are all of high importance for a surgeon wishingto immediately administer the suspension to a patient undergoingsurgery. Even after longer periods of shaking, the control microspheresinclude a fraction which float on the surface.

The method of the invention thus allows easier handling, improved dosagecontrol, and faster rehydration, all of high importance for a surgeon orinterventional radiologist wishing to immediately administer thesuspension to a patient undergoing surgery.

Example 2

The beads produced as in Example 1 are loaded with irinotecan loaded ata level of around 50 mg drug per ml beads. The lyophilisation cycle wasthe same as that used in example 1. The beads could easily be rehydratedupon addition of saline to the container under lower than atmosphericpressure, the beads quickly sinking and being capable of forming ahomogeneous suspension.

Example 3

Alginate microspheres are formed as follows. An aqueous solution of highG alginate (recovered as described in WO 00/09566 A) is cross-linked byspraying droplets of the 2% solution into a precipitation bathcomprising a solution of calcium ions, followed by collection of theformed microspheres. The micro spheres have an average size of 215 μm(standard deviations 3 μm). After cleaning, 0.2 ml of 2% alginatemicrosphere suspension is transferred into a vial. Excess liquid isdecanted, then 1.39 mls of 10.07 mg per ml aqueous doxorubicin solutionis added to the microspheres. The mixture is shaken overnight. Afterthis time, the loading capacity is determined by measuring theconcentration of a portion of decanted excess loading solution. Thisreveals a loading capacity of around 50 to 60 mg doxorubicin per ml ofbead suspension. Excess loading solution is removed and the bead slurrysubjected to freeze drying in the vial.

The freeze drying cycle is substantially as described in example 1. Atthe end of the cycle the vials are stoppered under the final vacuum.

When resuspended in 0.9 wt % saline, the beads rehydrated rapidly, andsank in the suspension.

What is claimed is:
 1. A method for formulating a dried packaged productsuitable for direct administration to an animal after rehydration toform a suspension comprising: i) providing particles of polymer matrixswollen with water and having absorbed therein a non-volatilebiologically active compound; ii) cooling the swollen articles from stepi) in a freezing step to a temperature below the freezing point forwater; iii) in a lyophilisation step subjecting the cooled particlesfrom step ii) to a reduced pressure at which ice sublimes for a periodduring which at least a portion of the absorbed ice sublimes and watervapour is removed; and iv) in a packaging step, packaging the driedparticles under reduced pressure and into a package that issubstantially airtight and has an interior under vacuum to form packagedparticles.
 2. A method according to claim 1, in which the said packagecomprises a vessel which is substantially rigid and has a mouth closedby a stopper and in which the pressure inside the package is less than0.95 bar.
 3. A method according to claim 3, in which the particles ofswollen polymer are contained in the vessel during the freezing andlyophilisation steps and in which the stopper is fitted into the mouthof the vessel in the packaging step.
 4. A method according to claim 1,in which the temperature to which the particles are cooled in thecooling step is less than −20° C.
 5. A method according to claim 1, inwhich the lyophilisation step is carried out at a temperature less than−20° C.
 6. A method according to claim 1, in which the pressure in thelyophilisation step is reduced to less than 100 mbar for.
 7. A methodaccording to claim 1, in which the polymer is a water-insoluble,pharmaceutically acceptable polymer.
 8. A method according to claim 7,in which the polymer is cross-linked.
 9. A method according to claim 7,in which the polymer is based on poly(vinyl alcohol).
 10. A methodaccording to claim 7, in which the polymer is formed by polymerisationof ethylenically unsaturated monomers.
 11. A method according to claim1, in which the particle sizes are selected such that upon rehydrationin 0.9 wt % saline at room temperature, the average particle size is inthe range 40 to 2000 μm.
 12. A method according to claim 1, in which theparticles are substantially spherical in shape.
 13. A method accordingto claim 1, in which the biologically active compound is selected fromanti-proliferatives, including anti-neoplastics, anti-migratories,immunosuppressants, analgesics, anti-inflammatories, anti-pyretics,anaesthetics and anti-bacterials.
 14. A method according to claim 13, inwhich the biologically active is an anti-neoplastic agent selected fromangiopeptin, statins, such as sandostatin, azacitidine, bleomycin andbleomycin sulphate, carboplatin, cisplatin, streptozoticin,capecitabine, vinorelbine, cyclosporin, cytabanine, dacarbazine,anthracyclines such as daunorubicin hydrochloride and doxorubicinhydrochloride, fluorouracil, haropiridol, gemcitabine, ifosfamide,methotrexate, mitoxantrone, banoxantrone, mitomycin, mustinehydrochloride, lomustine, carmustine/BCNU, meclorethamine, vincristine,vinblastine and cytosar/cytarabine, paclitaxel, docetaxel, rapamycin andderivatives, such as tyrphostin, tacrolimus, everolimus, biolimus,zotarolimus and RAD001, tetradecylselenoacetic acid, tetradecylthioacetic acid, ethylisopropylamiloride, antithrombin, aggrastat,cilostazol, clexane, clopidogrel, dipyridamole, persantine, integrillin,abciximab, trapidil, matrix metalloproteinase inhibitors such asbatimastat and marimastat, VEGF, carvedilol, estradiol and otherestrogens, L-arginine, nitric oxide donors, probucol, quinaprilat,thioctacid, telmisartan, zoledronate, and irinotecan.
 15. A method ofpreparing a pharmaceutical suspension for administration to an animal inwhich the product of a method according to claim 1, optionally after astorage period of 1 day to 10 years, is rehydrated by adding to thepackage of dried product a pharmaceutically acceptable sterile aqueousliquid, substantially without allowing ingress of air, and, optionally,a contrast medium, to form a suspension of swollen particles in theaqueous liquid.
 16. A method of treatment of an animal in which there isprepared a pharmaceutical suspension for administration to an animal inwhich the product of a method according to claim 1, optionally after astorage period of 1 day to 10 years, is rehydrated by adding to thepackage of dried product a pharmaceutically acceptable sterile aqueousliquid, substantially without allowing ingress of air, and, optionally,a contrast medium, to form a suspension of swollen particles in theaqueous liquid and the suspension is administered to an animal.
 17. Anairtight package containing, under vacuum, lyophilised particles ofwater-swellable water-insoluble biocompatible polymer into which isabsorbed a pharmaceutically acceptable biologically active compound, inwhich the particles are swellable in 0.9 wt % saline at room temperatureto sizes in the range 40 to 2000 μm.
 18. Package according to claim 17,in which the package is rigid.
 19. Package according to claim 18, inwhich the package is of glass closed with an airtight closure, includinga stopper penetrable by hypodermic needle.
 20. Package according toclaim 17, in which the polymer is a water-insoluble, pharmaceuticallyacceptable polymer.